Efforts to determine the nucleotide sequence of complete genomes, or a large portion thereof, have traditionally taken a so-called bottom-up approach, including preparing a library of random large DNA clones in yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1 or cosmids, followed by random subcloning in M13-like vectors without great reliance upon genome mapping. Among such systems, BACs are at present the preferred vector for maintaining large genomic DNA fragments. BACs are preferred because individual DNA fragments are maintained stably in a single-copy (SC) vector in the host cells, even after 100 or more generations of serial bacterial growth. In contrast, the DNA fragments cloned into YACs tend to be unstable and can yield chimeric clones. It is difficult to recover DNA clones from YACs in a pure form.
BAC (or pBAC) vectors typically accommodate inserts in the range of approximately 30 to 300 kilobase pairs. A widely used BAC vector, pBeloBac11, uses a complementation of the lacZ gene to distinguish insert-containing recombinant molecules from colonies carrying the BAC vector, by color. When a DNA fragment is cloned into the lacZ gene of pBeloBac11, insertional inactivation results in a white colony on X-Gal/IPTG plates after transformation. Kim, U-J et al., “Construction and Characterization of a Human Bacterial Artificial Chromosome Library,” Genomics 34:213-218 (1996). Thus, it is now possible to distinguish those colonies that contain BACs with DNA inserts from those that lack inserts. A similar prior vector, pBAC108L, lacked the ability to distinguish insert-containing BACs. Shizuya, H., “Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector,” P.N.A.S. U.S.A. 89:8794-8797 (1992).
Although these SC vectors are advantageously used to clone large genomic DNA fragments for subsequent analysis, especially sequence analysis, the single copy nature of these vectors is also a limitation in that large numbers of cells containing a BAC clone of interest must be grown to produce a sufficient quantity of DNA for subsequent analysis. It is, of course, possible to amplify portions of a BAC clone of interest using, for example, PCR, but simple amplification of the entire insert from a BAC vector has not previously been possible.
U.S. Pat. No. 5,874,259 discloses conditionally amplifiable BAC vectors into which large genomic DNA fragments can be inserted. The conditionally amplifiable BAC vectors contain, in addition to an origin of replication that maintains the vectors at one copy per cell, a conditional origin of replication (conditional ori) at which replication is initiated in response to a suitable signal in the host cell. After a genomic DNA fragment is inserted into a conditionally amplifiable BAC vector, a large amount of the genomic DNA fragment can be obtained through inducing the replication of the BAC vector from its conditional ori. The vectors in U.S. Pat. No. 5,874,259 can optionally contain a pair of excision-mediating sites (EMS) flanking the conditional ori and a site into which a genomic DNA fragment can be cloned. In this case, the nucleic acid between the EMS can be excised to create a circular plasmid that comprises the genomic fragment insert and can replicate when the conditional ori is induced.
SC BAC vectors, and BAC libraries created using SC BAC vectors (such as pBeloBac11 and pBAC108L), are known. U.S. Pat. No. 5,874,259 discloses methods and vectors for constructing a conditionally amplifiable BAC library. A method for converting existing insert-containing SC BAC clones to conditionally amplifiable BAC clones is needed in the art.